BIOMARKERS FOR BREAST CANCER

Abstract

) peptides have been discovered that are indicative of breast cancer. Evaluating patient samples for the presence of such LMW peptides is an effective means of detecting breast cancer and monitoring the progression of the disease, for example during treatment. The LMW peptides are particularly useful in detecting breast cancer during its early stages.

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001]This application claims priority to U.S. Provisional Application No. 60/855,378, filed Oct. 31, 2006, which is hereby incorporated by reference.

BACKGROUND

[0002]Breast cancer is the most frequent neoplasm and the leading cause of cancer mortality in women worldwide. According to estimates, approximately 41,000 women in the United States and 130,000 women in the European Union die for breast cancer yearly. Mammographic screening has been widespread for the past twenty years and shown to reduce breast cancer mortality by 20-35% in women aged 40 to 69 years.

[0003]Mammography has a variety of short-comings, however. For example, according to the current guidelines, some women who develop breast cancer are "too young" to start regular mammograms. Also, less than half of eligible women get mammograms regularly, and the predictive value of mammography declines in cohorts of patients with denser breast tissue and smaller lesions. Furthermore, mammography is not effective in detecting early stages of breast cancer.

[0004]Needless to say, early detection is of paramount importance in reducing mortality from this major public health burden. Detection of breast cancer at the earliest stages results in a much greater favorable outcome, with 10-year disease-free survival rate as high as 98% in patients with pT1a,bN0M0 tumors (measuring 1 cm or less, with disease-free axillary lymph nodes and no distant metastasis). Thus, the potential for enhancing treatment by providing an early diagnosis has driven a search for better diagnostic tools.

[0005]Some biomarker genes and proteins, such as BRCA1, BRCA2 and Her-2/neu, have been identified and developed into tools for genetic screening. The advantages and limitations of these detection approaches have been discussed in the literature. See, e.g. Ponzone et al., Eur. J. Cancer 34(7): 966-967, 1998; Bradbury, Lancet Oncol. 3: 2, 2002; Ross et al., Expert Rev. Mol. Diagn. 3(5): 573-585, 2003.

[0006]A need exists, however, for additional biomarkers useful for detecting breast cancer, and in particular biomarkers that can detect early stages of the disease.

SUMMARY

[0007]In one embodiment, a method for detecting breast cancer in a patient comprises obtaining a biological sample from the patient and evaluating the sample or a fraction of the sample for the presence of at least one biomarker selected from the group of peptides having the sequence of SEQ ID NOs: 1-217, wherein the presence of said at least one biomarker is indicative of breast cancer. In one aspect, the methods involve evaluating the sample for the presence of a biomarker selected from the group of peptides having the amino acid sequence of SEQ ID NOs: 132-217. In another, the methods comprise evaluating the sample for the presence of peptides having the amino acid sequence of SEQ ID NOs: 132, 139, 141 and 148. In one aspect, the breast cancer is in early stage, such as stage T1a. The biological sample can be, for example, blood, serum or plasma. In one embodiment, the evaluation step comprises assays such as mass spectrometry, an immunoassay such as ELISA, immunomass spectrometry or suspension bead array.

[0008]In another embodiment, the method further comprises, prior to the evaluation step, harvesting low molecular weight peptides from the biological sample to generate at least one fraction comprising the peptides. In one embodiment, the size of the low molecular weight peptides is less than 50 KDa, preferably less than 25 KDa, and more preferably less than 15 KDa. In another aspect, the method also comprises digesting the low molecular weight peptides. Such digestion can be accomplished using enzymatic or chemical means. In one example, trypsin can be used to digest the peptides.

[0009]In another aspect, a method for monitoring the progression of breast cancer in a patient comprises (i) obtaining a biological sample from the patient, (ii) evaluating the sample or a fraction of the sample for the presence of at least one biomarker selected from the group of peptides having the sequences of SEQ ID NOs: 1-217, wherein the presence of said at least one biomarker is indicative of breast cancer, and optionally, repeating steps (i) and (ii) as necessary. In one aspect, the methods involve evaluating the sample for the presence of a biomarker selected from the group of peptides having the amino acid sequence of SEQ ID NOs: 132-217. In another, the methods comprise evaluating the sample for the presence of peptides having the amino acid sequence of SEQ ID NOs: 132, 139, 141 and 148. In one embodiment, the method further comprises a step of harvesting low molecular weight peptides from the sample to generate at least one fraction comprising the peptides.

[0010]In other aspects, the invention relates to antibodies specific for identified biomarkers for breast cancer, as well as kits for detecting breast cancer in a patient, comprising at least one such antibody.

[0011]Other objects, features and advantages will become apparent from the following detailed description. The detailed description and specific examples are given for illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Further, the examples demonstrate the principle of the invention and cannot be expected to specifically illustrate the application of this invention to all the examples where it will be obviously useful to those skilled in the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 provides a CID Spectrum of peptide "CFVESLSSVETLK" from CDK4 protein identified only in LMW of breast cancer serum (accession number Q96BE9, amino acid residues 90 -102).

[0013]FIG. 2 provides a CID Spectrum of peptide "MVFHITTGSQEFDK" from meiotic recombination protein DMC1/LIM15 homolog identified only in LMW of breast cancer serum (accession number Q14565, amino acid residues 97-110).

[0014]FIG. 3 provides a CID Spectrum of peptide "EVGNLLLENSQLLETK" from C-jun-amino-terminal kinase interacting protein 3 identified only in LMW of breast cancer serum (accession number Q9UPT6, amino acid residues 417-432).

DETAILED DESCRIPTION

[0015]Low molecular weight (LMW) peptides have been discovered that are indicative of breast cancer. Evaluating patient samples for the presence of such LMW peptides is an effective means of detecting breast cancer and monitoring the progression of the disease, for example during treatment. The LMW peptides are particularly useful in detecting breast cancer during its earliest stages, such as stage I.

[0016]The LMW peptides, or biomarkers, can be detected using a variety of methods known in the art. For example, antibodies can be utilized in immunoassays to detect the presence of a biomarker. Exemplary immunoassays include, e.g., ELISA, radioimmunoassay, immunofluorescent assay, "sandwich" immunoassay, western blot, immunoprecipitation assay and immunoelectrophoresis assays. Furthermore, methods involving beads, microbeads, arrays, microarrays, etc. can be applied in detecting the LMW peptides. Exemplary assays include, but are not limited to, suspension bead assays (Schwenk et al., "Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics," Mol. Cell Proteomics, 6(1): 125-132 (2007)), antibody microarrays (Borrebaeck et al., "High-throughput proteomics using antibody microarrays: an update," Expert Rev. Mol. Diagn. 7(5): 673-686 (2007)), aptamer arrays (Walter et al., "High-throughput protein arrays: prospects for molecular diagnostics," Trends Mol. Med. 8(6): 250-253 (2002)), affybody arrays (Renberg et al., "Affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats," J. Proteome Res. 6(1): 171-179 (2007)), and reverse phase arrays (VanMeter et al., "Reverse-phase protein microarrays: application to biomarker discorvery and translational medicine," Expert Rev. Mol. Diagn. 7(5): 625-633 (2007)). All these publications are incorporated herein by reference.

[0017]In another example, the inventive biomarkers can be detected using mass spectrometry (MS). One example of this approach is tandem mass spectrometry (MS/MS), which involves multiple steps of mass selection or analysis, usually separated by some form of fragmentation. Most such assays use electrospray ionization followed by two stages of mass selection: a first stage (MS 1) selecting the mass of the intact analyte (parent ion) and, after fragmentation of the parent by collision with gas atoms, a second stage (MS2) selecting a specific fragment of the parent, collectively generating a selected reaction monitoring assay. In one embodiment, collision-induced dissociation is used to generate a set of fragments from a specific peptide ion. The fragmentation process primarily gives rise to cleavage products that break along peptide bonds. Because of the simplicity in fragmentation, the observed fragment masses can be compared to a database of predicted masses for known peptide sequences. A number of different algorithmic approaches have been described to identify peptides and proteins from tandem mass spectrometry (MS/MS) data, including peptide fragment fingerprinting (SEQUEST, MASCOT, OMSSA and X!Tandem), peptide de novo sequencing (PEAKS, LuteFisk and Sherenga) and sequence tag based searching (SPIDER, GutenTAG).

[0018]10018] Likewise, multiple reaction monitoring (MRM) can be used to identify the inventive biomarkers in patient samples. This technique applies the MS/MS approach to, for example, tryptic digests of the input sample, followed by selected ion partitioning and sampling using MS to objectify and discreetize the analyte if interest by following the exact m/z ion of the tryptic fragment that represents the analyte. Such an approach can be performed in multiplex so that multiple ions can be measured at once, providing an antibody-free method for analyte measurement. See, e.g. Andersen et al., "Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins," Molecular & Cellular Proteomics, 5.4: 573-588 (2006); Whiteaker et al., "Integrated pipeline for mass spectrometry-based discorvery and confirmation of biomarkers demonstrated in a mouse model of breast cancer," J. Proteome Res. 6(10): 3962-75 (2007). Both publications are incorporated herein by reference in their entirety.

[0019]In another example, the inventive biomarkers can be detected using nanoflow reverse-phase liquid chromatography-tandem mass spectrometry. See, e.g., Domon B, Aebersold R. "Mass spectrometry and protein analysis." Science, 312(5771):212-7(2006), which is incorporated herein by reference in its entirety. Using this approach, experimentalists obtain peptide fragments, usually by trypsin digest, and generate mass spectrograms of the fragments, which are then compared to a database, such as SEQUEST, for protein identification.

[0020]In another aspect, the inventive biomarkers can be detected using immuno-mass spectrometry. See, e.g., Liotta L et al. "Serum peptidome for cancer detection: spinning biologic trash into diagnostic gold." J Clin Invest.,116(1):26-30 (2006); Nedelkov, "Mass spectrometry-based immunoassays for the next phase of clinical applications," Expert Rev. Proteomics, 3(6): 631-640 (2006), which are incorporated herein by reference. Immuno-mass spectrometry provides a means for rapidly determining the exact size and identity of a peptide biomarker isoform present within a patient sample. When developed as a high throughput diagnostic assay, a drop of patient's blood, serum or plasma can be applied to a high density matrix of microcolumns or microwells filled with a composite substratum containing immobilized polyclonal antibodies, directed against the peptide marker. All isoforms of the peptide that contain the epitope are captured. The captured population of analytes including the analyte fragments are eluted and analyzed directly by a mass spectrometer such as MALDI-TOF MS. The presence of the specific peptide biomarker at its exact mass/charge (m/z) location would be used as a diagnostic test result. The analysis can be performed rapidly by simple software that determines if a series of ion peaks are present at defined m/z locations.

[0021]In yet another example, the inventive biomarkers can be detected using standard immunoassay-based approaches whereby fragment specific antibodies are used to measure and record the presence of the diagnostic fragments. See, e.g., Naya et al. "Evaluation of precursor prostate-specific antigen isoform ratios in the detection of prostate cancer." Urol Oncol. 23(1):16-21 (2005). Moreover, additional immunoassays are well known to those skilled in the field, such as ELISAs (Maeda et al., "Blood tests for asbestos-related mesothelioma," Oncology 71: 26-31 (2006)), microfluidic ELISA (Lee et al., "Microfluidic enzyme-linked immunosorbent assay technology," Adv. Clin. Chem. 42: 255-259 (2006)), nanocantilever immunoassays (Kurosawa et al., "Quartz crystal microbalance immunosensors for environmental monitoring," Biosens Bioelectron, 22(4): 473-481 (2006)), and plasmon resonance immunoassays (Nedelkov, "Development of surface Plasmon resonance mass spectrometry array platform," Anal. Chem. 79(15): 5987-5990 (2007)). All of these publications are incorporated herein by reference.

[0022]In a further example, the biomarkers can be detected using electrochemical approaches. See, e.g., Lin et al., "Electrochemical immunosensor for carcinoembryonic antigen based on antigen immobilization in gold nanoparticles modified chitosan membrane," Anal. Sci. 23(9): 1059-1063 (2007).

[0023]In one embodiment, the LMW peptides are harvested from a biological sample prior to the evaluation step. For example, 100 μl of serum can be mixed with 2× SDS-PAGE Laemmli Buffer (containing 200 mM DTT), boiled for 10 minutes, and loaded on Prep Cell (Model 491 Prep Cell, Bio-Rad Laboratories, CA) comprising a 5 cm length 10% acrylamide gel. Electrophoresis is performed under a constant voltage of 250V. Immediately after the bromophenol blue indicator dye is eluted from the system, LMW peptides and proteins migrate out of the gel and are trapped in a dialysis membrane in the elution chamber. These molecules can be eluted at a flow rate of 400 ml/l min by a buffer with the same composition of the Tris-Glycine running buffer and collected for 10 minutes in one fraction.

[0024]Alternatively, LMW peptides can be harvested using from a sample using a capture-particle that comprises a molecular sieve portion and an analyte binding portion as described in U.S. patent application Ser. No. 11/527,727, filed Sep. 27, 2006, which is incorporated herein by reference in its entirety. Briefly, either the molecular sieve portion or the analyte binding portion or both comprise a cross-linked region having modified porosity, or pore dimensions sufficient to exclude high molecular weight molecules.

[0025]In another embodiment, the LMW peptides are digested prior to detection, so as to reduce the size of the peptides. Such digestion can be carried out using standard methods well known in the field. Exemplary treatments, include but are not limited to, enzymatic and chemical treatments. Such treatments can yield partial as well as complete digestions. One example of an enzymatic treatment is a trypsin digestion.

[0026]The inventive biomarkers are particularly useful in detecting breast cancer during its early stages, i.e., prior to metastasis and large tumor volume (e.g. greater than 2 cm).

[0027]Antibodies specific for the inventive biomarkers can be produced readily using well known methods in the art. (See, J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning, a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, pp. 18.7-18.18, 1989) For example, the inventive biomarkers can be prepared readily using an automated peptide synthesizer. Next, injection of an immunogen, such as (peptide)_n-KLH (n=1-30) in complete Freund's adjuvant, followed by two subsequent injections of the same immunogen suspended in incomplete Freund's adjuvant into immunocompetent animals, is followed three days after an i.v. boost of antigen, by spleen cell harvesting. Harvested spleen cells are then fused with Sp2/0-Ag14 myeloma cells and culture supernatants of the resulting clones analyzed for anti-peptide reactivity using a direct-binding ELISA. Fine specificity of generated antibodies can be detected by using peptide fragments of the original immunogen.

[0028]In certain embodiments, one or more antibodies directed to the inventive biomarkers is provided in a kit, for use in a diagnostic method. Such kits also can comprise reagents, instructions and other products for performing the diagnostic method.

[0029]The detailed description of the present invention is provided below by the following example, which is illustrative only and not limiting the invention in any way.

Examples

Example 1

Identification of Biomarkers for Breast Cancer Using LTQ

[0030]Blood Collection and Serum Preparation

[0031]Blood samples were drawn from patients before the mammography screening under full Institutional Review Board approval and patient's consent. Specimens were collected in red-top Vacutainer Tubes and allowed to clot for 1 hour on ice, followed by centrifugation at 4° C. for 10 minutes at 2000 g. The serum supernatant was divided in aliquots and stored at -80° C. until needed. 10 serum samples with negative outcome were pooled in a single control group. 10 serum samples from patients with a diagnosed T1a stage breast cancer were pooled in a single disease group. Each experiment has been performed using 3 different aliquots from the same pool, both, for the control and for the disease group.

[0032]Low Molecular Weight (LMW) Protein Harvesting by Continuous Elution Electrophoresis

[0033]100 μl of serum was mixed with 2× SDS-PAGE Laemmli Buffer (containing 200 mM DTT), boiled for 10 minutes, and loaded on Prep Cell (Model 491 Prep Cell, Bio-Rad Laboratories, CA) in which 5 cm length 10% acrylamide gel was polymerized. Electrophoresis was performed under a constant voltage of 250V. Immediately after the bromophenol blue indicator dye was eluted from the system, LMW peptides and proteins migrate out of the gel and are trapped in a dialysis membrane in the elution chamber. These molecules were eluted at a flow rate of 400 μl min by a buffer with the same composition of the Tris-Glycine running buffer and collected for 10 minutes in one fraction.

[0034]SDS Removal from the Prep Cell Fractions

[0035]LMW fractions obtained by the Prep Cell were processed using a commercially available ion-exchange matrix (Proteo Spin Detergent Clean-Up Micro Kit, Norgen Biotek Corporation, Canada) following protocols outlined by the manufacturer for both acidic and basic proteins, resulting in a final volume of 55 μl.

[0036]Nanoflow Reversed-Phase Liquid Chromatography-Tandem MS (nanoRPLC-MS/MS)

[0037]The SDS-free LMW fractions obtained from the described procedure were analyzed by traditional bottom-up MS approaches. This was accomplished by treating the samples by reduction using 20 mM DTT, followed by alkylation using 100 mM iodoacetamide and lastly, trypsin digestion (Promega, WI) at 37° C. overnight in 50 mM ammonium bicarbonate in the presence of 1M urea in a final volume of 200 pl. Tryptic peptides were desalted by μC₁₈ Zip Tip (Millipore, MA) and analyzed by reversed-phase liquid chromatography nanospray tandem mass spectrometry using a linear ion-trap mass spectrometer (LTQ, ThermoElectron, San Jose, Calif.). Reverse phase column was slurry-packed in-house with 5 μm, 200 Å pore size C₁₈ resin (Michrom BioResources, CA) in 100 μm i.d.×10 cm long fused silica capillary (Polymicro Technologies, Phoenix, Ariz.) with a laser-pulled tip. After sample injection, the column was washed for 5 min with mobile phase A (0.4% acetic acid, 0.005% heptafluorobutyric acid) and peptides were eluted using a linear gradient of 0% mobile phase B (0.4% acetic acid, 0.005% heptafluorobutyric acid, 80% acetonitrile) to 50% mobile phase B in 30 min at 250 nl/min, then to 100% B in an additional 5 min. The LTQ mass spectrometer was operated in a data-dependent mode in which each full MS scan was followed by five MS/MS scans where the five most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35%.

[0038]Bioinformatic Analysis

[0039]Tandem mass spectra were matched against Swiss-Prot human protein database through SEQUEST algorithm incorporated in Bioworks software (version 3.2, Thermo Electron) using tryptic cleavage constraints and static cysteine alkylation by iodoacetamide. For a peptide to be considered legitimately identified, it had to achieve Delta Cn value above 0.1, cross correlation scores of 1.5 for [M+H]¹+, 2.0 for [M+2H]²+, 2.5 for [M+3H]³+, and a probability cut-off for randomized identification of p<0.01.

[0040]The results are provided in Table 1. In short, 131 peptides were identified as biomarkers that correlate to the disease state. Thus, evaluating patient samples for the presence of one or more of these biomarkers will provide a useful method for detecting breast cancer.

TABLE-US-00001 TABLE 1 SEQ ID Residue Proteins P (pro) MW Accession Amino acid sequence NO. number NOTC1_HUMAN (P46531) Neurogenic 2.96E-03 272372.2 P46531 QWTQQHLDAADLR 1 1843-1855 locus notch homolog protein 1 precursor (Notch 1) (hN1) (Translocat Q4G171 (Q4G171) CASC1 protein 4.95E-03 79322.33 Q4G171 QASTLADLDSGNMEK 2 254-268 Q69YN4 (Q69YN4) Hypothetical protein 4.77E-04 201574 Q69YN4 NLRFEINCIPNLIEYVK 3 898-914 DKFZp686C1522 Q8N3Y7 (Q8N3Y7) Epidermal retinal 6.86E-03 34072.78 Q8N3Y7 KEVGDVSILINNAGIVTGK 4 114-132 dehydrogenase 2 PSD1_HUMAN (Q99460) 26S proteasome 4.79E-03 105768.7 Q99460 NNNTDLMILKNTKDAVR 5 345-361 non-ATPase regulatory subunit 1 (26S proteasome regulatory subun KCNH6_HUMAN (Q9H252) Potassium 7.88E-04 109855.1 Q9H252 GSIEILRDDVVVAILGK 6 637-653 voltage-gated channel subfamily H member 6 (Voltage-gated potassium WDR36_HUMAN (Q8NI36) WD-repeat 6.93E-03 105255.2 Q8NI36 TASALFAGF 7 65-88 protein 36 (T-cell activation WD repeat RALGLFSNDIPHVVR protein) (TA-WDRP) FLRT2_HUMAN (O43155) Leucine-rich 1.92E-03 74001.84 O43155 ERVTPPISER 8 415-424 repeat transmembrane protein FLRT2 precursor (Fibronectin-like do PCNT2_HUMAN (O95613) Pericentrin-2 1.16E-03 377847.9 O95613 AAGSDADHLREQQR 9 2960-2973 (Pericentrin B) (Kendrin) Q15813 (Q15813) Beta-tubulin cofactor E 5.90E-05 59309.08 Q15813 PNKVNFGTDFLTAIKNR 10 72-88 (Tubulin-specific chaperone e) CAN12_HUMAN (Q6ZSI9) Calpain-12 (EC 4.58E-03 80985.57 Q6ZSI9 TPKCTVLLSLIQR 11 421-433 3.4.22.--) Q96BB9 (Q96BB9) IGHM protein 1.14E-03 64998.24 Q96BB9 DTLYLQMNSLR 12 96-106 SNX23_HUMAN (Q96L93) Kinesin-like 4.35E-04 151916.8 Q96L93 TLKLKYAELAALEFPPK 13 1230-1246 motor protein C20orf23 (Sorting nexin 23) E41LB_HUMAN (Q9H329) Band 4.1-like 9.77E-03 100657.1 Q9H329 SPAQAELSYLNKAK 14 240-253 protein 4B (EHM2 protein) (FERM- containing protein CG1) EPHA5_HUMAN (P54756) Ephrin type-A 4.48E-03 114710.3 P54756 RLGVTLVGHQK 15 1007-1017 receptor 5 precursor (EC 2.7.1.112) (Tyrosine-protein kinase rec PSA7L_HUMAN (Q8TAA3) Proteasome 2.43E-03 28540.14 Q8TAA3 EVELYVTEIEKEKEEAEK 16 232-249 subunit alpha type 7-like (EC 3.4.25.1) Q9BVV2 (Q9BVV2) Hypothetical protein 5.03E-03 36503.15 Q9BVV2 DSLTLHTKPEPLEG 17 298-318 MGC5356 (OTTHUMP00000031567) PALSHSV MRGBP_HUMAN (Q9NV56) MRG-binding 4.77E-03 22402.99 Q9NV56 EDVDPHNGADDVFSS 18 132-156 protein SGSLGKASEK JIP3_HUMAN (Q9UPT6) C-jun-amino- 3.33E-03 146961.6 Q9UPT6 EVGNLLLENSQLLETK 19 417-432 terminal kinase interacting protein 3 (JNK-interacting protein 3) ( LG3BP_HUMAN (Q08380) Galectin-3 2.47E-03 65289.4 Q08380 SDLAVPSELALLK 20 311-323 binding protein precursor (Lectin galactoside-binding soluble 3 bin SNX17_HUMAN (Q15036) Sorting nexin- 1.58E-03 52868.24 Q15036 SPPLLESPDATRESMVKLSSK 21 415-435 17 Q8N8J8 (Q8N8J8) Hypothetical protein 8.60E-03 35709.23 Q8N8J8 EACIVEALGIQTLTNQK 22 257-273 FLJ39369 Q9NU63 (Q9NU63) Chromosome 6 open 6.64E-04 51869.71 Q9NU63 MAAGEPRSLLFFQK 23 1-14 reading frame 40 (Fragment) DMC1_HUMAN (Q14565) Meiotic 3.02E-03 37657.33 Q14565 MVFHITTGSQEFDK 24 97-110 recombination protein DMC1/LIM15 homolog THAP4_HUMAN (Q8WY91) THAP domain 8.64E-03 62850.72 Q8WY91 FIGSLHSYSFSSKHTR 25 231-246 protein 4 ROBO4_HUMAN (Q8WZ75) Roundabout 1.83E-04 107390.4 Q8WZ75 PAVWLSWK 26 262-285 homolog 4 precursor (Magic roundabout) VSGPAAPAQSYTALFR HTRA1_HUMAN (Q92743) Serine 6.04E-03 51254.68 Q92743 YIGIRMMSLTSSK 27 382-394 protease HTRA1 precursor (EC 3.4.21.--) (L56) TLR8_HUMAN (Q9NR97) Toll-like receptor 8.10E-03 119752.5 Q9NR97 NLYLAWNCYFNKVCEK 28 174-189 8 precursor HXA3_HUMAN (O43365) Homeobox 5.13E-03 46339.7 O43365 VEMANLLNLTERQIK 29 222-236 protein Hox-A3 (Hox-1E) Q6ZR27 (Q6ZR27) Hypothetical protein 6.97E-03 54430.06 Q6ZR27 LVEVIPEGAMLRLG 30 208-237 FLJ46705 MTNPPYILEHLEEMAK Q6ZRS3 (Q6ZRS3) Hypothetical protein 6.45E-04 17827.01 Q6ZRS3 PAASPNTTSSRGQTV 31 23-46 FLJ46148 HPPCSSKLR Q8TAM1 (Q8TAM1) Hypothetical protein 4.40E-03 73940.78 Q8TAM1 NRLTDYYEPLLKN 32 465-485 FLJ23560 NSTAYSTR BCL9_HUMAN (O00512) B-cell lymphoma 9.09E-03 149218.7 O00512 FAMPSSTPLYHDAIK 33 1016-1030 9 protein (Bcl-9) (Legless homolog) AKAP3_HUMAN (O75969) A-kinase 9.40E-05 94676.41 O75969 SCDASLAELGDDKSGDASR 34 687-705 anchor protein 3 (Protein kinase A anchoring protein 3) (PRKA3) (A-ki O95973 (O95973) VH4 heavy chain 1.29E-05 16305.14 O95973 VTISVDTSK 35 88-96 variable region precursor (Fragment) Q5SZH6 (Q5SZH6) Novel protein 6.74E-04 53734.77 Q5SZH6 MDINTYNNQLHLQR 36 1-14 Q9UL71 (Q9UL71) Myosin-reactive 9.27E-03 13145.42 Q9UL71 AEDTALYYCAK 37 88-98 immunoglobulin heavy chain variable region (Fragment) MFA3L_HUMAN (O75121) Microfibrillar- 1.22E-03 45351.02 O75121 SHLTVCFLPSVPF 38 6-29 associated protein 3-like precursor LILVSTLATAK (Protein kinase NYD-SP9) GOGA4_HUMAN (Q13439) Golgi 7.07E-04 260978.6 Q13439 ELSENINAVTLMKEELKEK 39 1347-1365 autoantigen, golgin subfamily A member 4 (Trans-Golgi p230) (256 kDa gol AUTS2_HUMAN (Q8WXX7) Autism 4.77E-03 138897.1 Q8WXX7 PGQNSCRDSDSES 40 177-199 susceptibility gene 2 protein ASGESKGFHR CELR1_HUMAN (Q9NYQ6) Cadherin EGF 6.96E-03 329276.7 Q9NYQ6 DANSVITYQLTGGNTR 41 715-730 LAG seven-pass G-type receptor 1 precursor (Flamingo homolog 2) ( WRN_HUMAN (Q14191) Werner 1.41E-03 162390.4 Q14191 DEIQCVIATIAFGMGINKADIR 42 813-834 syndrome helicase Q5R329 (Q5R329) Testicular soluble 9.72E-03 187025.2 Q5R329 AVIKNRNTTYIV 43 685-708 adenylyl cyclase (SAC) IGAVQPNDISNK Q5TF21 (Q5TF21) 1.66E-03 103136.2 Q5TF21 VMQLQYENRVLMSNMQRY 44 719-745 OTTHUMP00000017175 DLASHLGIR Q9H0R6 (Q9H0R6) Hypothetical protein 3.65E-03 57432.23 Q9H0R6 GRILSGNFFLLKENYENYFVK 45 375-395 DKFZp564C1278 TENX_HUMAN (P22105) Tenascin-X 8.10E-03 464165.9 P22105 DRDGRPQVVR 46 2203-2212 precursor (TN-X) (Hexabrachion-like protein) DOCK1_HUMAN (Q14185) Dedicator of 2.23E-03 215237.7 Q14185 NVEVTVSVYDEDGKR 47 447-461 cytokinesis protein 1 (180 kDa protein downstream of CRK) (DOCK18 DKK4_HUMAN (Q9UBT3) Dickkopf-related 6.46E-03 24859.18 Q9UBT3 KGQEGESC 48 138-159 protein 4 precursor (Dkk-4) LRTFDCGPGLCCAR (Dickkopf-4) (hDkk-4) [Contains: D RPC1_HUMAN (O14802) DNA-directed 3.06E-03 155648.3 O14802 TCCHIMLSQEEK 49 111-122 RNA polymerase III largest subunit (EC 2.7.7.6) (RPC155) (RPC1) FA10_HUMAN (P00742) Coagulation 8.74E-03 54696.55 P00742 CKDGLGEYTCTCLEGFEGK 50 101-119 factor X precursor (EC 3.4.21.6) (Stuart factor) (Stuart-Prower fac XCL1_HUMAN (P47992) Lymphotactin 3.14E-03 12508.7 P47992 AVIFITKRGLK 51 57-67 precursor (XCL1) (Cytokine SCM-1) (ATAC) (Lymphotaxin) (SCM-1-alph Q4J6C4 (Q4J6C4) Prolyl endopeptidase- 3.44E-03 76692.8 Q4J6C4 QENEKPLPENMDAFEKVR 52 80-97 like variant E Q6P387 (Q6P387) C16orf46 protein 1.20E-03 42723.96 Q6P387 AKEFIIGTGWEEAVQGWGR 53 58-76 CI068_HUMAN (Q8N4H0) Protein C9orf68 2.80E-04 45029.9 Q8N4H0 LPKGMQARAPSQYSTR 54 178-193 ANKR6_HUMAN (Q9Y2G4) Ankyrin repeat 7.67E-03 75675.76 Q9Y2G4 EEAREEFLSASPEPR 55 283-297 domain protein 6 DKC1_HUMAN (O60832) H/ACA 2.91E-03 57506.79 O60832 LDTSQWPLLLK 56 46-56 ribonucleoprotein complex subunit 4 (EC 5.4.99.--) (Dyskerin) (Nucleolar p

FIBB_HUMAN (P02675) Fibrinogen beta 4.12E-08 55892.23 P02675 EEAPSLRPAPPPISGGGYR 57 54-72 chain precursor [Contains: Fibrinopeptide B] K1C18_HUMAN (P05783) Keratin, type I 5.18E-05 47897.57 P05783 DWSHYFKIIEDLRA 58 124-148 cytoskeletal 18 (Cytokeratin-18) QIFANTVDNAR (CK-18) (Keratin-18) (K18) Q6P1M6 (Q6P1M6) Insulin-like growth 6.24E-03 31653.76 Q6P1M6 FLNVLSPR 59 226-233 factor binding protein 3 WDR9_HUMAN (Q9NSI6) WD-repeat 5.21E-03 257060.4 Q9NSI6 QNCKGDSQPNK 60 1442-1452 protein 9 NCOR2_HUMAN (Q9Y618) Nuclear 7.54E-04 273863.1 Q9Y618 VVTLAQHISEVITQDYTR 61 2132-2149 receptor corepressor 2 (N-CoR2) (Silencing mediator of retinoic acid a ACTN1_HUMAN (P12814) Alpha-actinin 1 2.98E-03 102992.7 P12814 ICDQWDNLGALTQKRR 62 479-494 (Alpha-actinin cytoskeletal isoform) (Non-muscle alpha-actinin LAMB3_HUMAN (Q13751) Laminin beta-3 7.23E-03 129488.5 Q13751 LGQSSMLGEQGARIQSVK 63 1092-1109 chain precursor (Laminin 5 beta 3) (Laminin B1k chain) (Kalinin RYR3_HUMAN (Q15413) Ryanodine 7.53E-03 551577.3 Q15413 SCQSGEDEEEDEDKEKTFEEK 64 3587-3607 receptor 3 (Brain-type ryanodine receptor) (RyR3) (RYR-3) (Brain ryan MK06_HUMAN (Q16659) Mitogen- 6.39E-03 82628.7 Q16659 ALSDVTDEEEVQVDPRK 65 384-400 activated protein kinase 6 (EC 2.7.1.37) (Extracellular signal-regulate OR5DI_HUMAN (Q8NGL1) Olfactory 4.67E-03 35324.45 Q8NGL1 DVKDTVTEILDTKVFSY 66 297-313 receptor 5D18 Q96BE9 (Q96BE9) CDK4 protein 4.81E-03 11993.24 Q96BE9 CFVESLSSVETLK 67 90-102 Q9C063 (Q9C063) LYST-interacting 3.34E-03 25900.14 Q9C063 PCWELKKIMILK 68 167-178 protein LIP5 (Fragment) Q9HBL8 (Q9HBL8) HSCARG 6.57E-04 33323.32 Q9HBL8 DIGVPMTSVRLPCYF 69 142-167 ENLLSHFLPQK RRBP1_HUMAN (Q9P2E9) Ribosome- 1.65E-03 152380 Q9P2E9 ADSVANQGTKVEGITNQGKK 70 550-569 binding protein 1 (Ribosome receptor protein) (180 kDa ribosome recep HV3H_HUMAN (P01769) Ig heavy chain 3.23E-06 13157.39 P01769 AENTAVYYCAR 71 88-98 V-III region GA SEM3F_HUMAN (Q13275) Semaphorin-3F 1.27E-03 88325.28 Q13275 EPLIIHWAAS 72 88-109 precursor (Semaphorin IV) (Sema IV) PQRIEECVLSGK (Sema III/F) TXND2_HUMAN (Q86VQ3) Thioredoxin 1.47E-03 60424.09 Q86VQ3 MDVDKELGMESVK 73 1-13 domain-containing protein 2 (Spermatid- specific thioredoxin-1) (Sp Q8N9P0 (Q8N9P0) Hypothetical protein 3.11E-03 25142.83 Q8N9P0 DVAGARGAPPAWGQAPSPRR 74 178-197 FLJ36797 Q96CS4 (Q96CS4) Zinc finger protein 4.07E-03 56870.35 Q96CS4 ETYGHLGALGCAGPK 75 60-74 HIT-39 (Hypothetical protein FLJ90415) Q96MT7 (Q96MT7) Hypothetical protein 2.25E-03 111658.3 Q96MT7 KKILDADIQLK 76 558-568 FLJ31910 Q96PE2 (Q96PE2) Tumor endothelial 6.84E-04 221533.1 Q96PE2 PKMLVISGGDGYED 77 2022-2051 marker 4 TVGRFRLSSGGGSSSE ICAM5_HUMAN (Q9UMF0) Intercellular 7.47E-03 97270.1 Q9UMF0 EPETQPVCFFR 78 92-102 adhesion molecule 5 precursor (ICAM-5) (Telencephalin) AP4B1_HUMAN (Q9Y6B7) Adapter-related 9.85E-03 83207.77 Q9Y6B7 GPLLAACSSES 79 279-301 protein complex 4 beta 1 subunit (Beta RELCFVALCHVR subunit of AP-4) (AP-4 CBPD_HUMAN (O75976) 3.19E-03 152818.8 O75976 GASSSTNDASVPTTKEFETLIK 80 886-907 Carboxypeptidase D precursor (EC 3.4.17.22) (Metallocarboxypeptidase D) (gp180) TRY1_HUMAN (P07477) Trypsin I 2.00E-03 26541.09 P07477 NKPGVYTKVYNYVK 81 224-237 precursor (EC 3.4.21.4) (Cationic trypsinogen) S61A1_HUMAN (P61619) Protein 4.32E-04 52099.53 P61619 GTLMELGISPIVT 82 73-97 transport protein Sec61 alpha subunit SGLIMQLLAGAK isoform 1 (Sec61 alpha-1) AT8B2_HUMAN (P98198) Probable 5.19E-03 137351.9 P98198 ENKFPLSNQNMLLR 83 238-251 phospholipid-transporting ATPase ID (EC 3.6.3.1) (ATPase class I type Q6PIZ8 (Q6PIZ8) TRAV20 protein 2.83E-03 30530.23 Q6PIZ8 GRGSQGNLIFGKGTK 84 113-127 Q96MY8 (Q96MY8) Hypothetical protein 2.56E-03 55323.95 Q96MY8 ISSTSTDR 85 177-184 FLJ31695 TRIM8_HUMAN (Q9BZR9) Tripartite motif 5.83E-03 61449.34 Q9BZR9 QTVEVLDK 86 249-256 protein 8 (RING finger protein 27) (Glioblastoma-expressed RI Q9UCY0 (Q9UCY0) OVCA1 = CANDIDATE 9.18E-03 48820.3 Q9UCY0 QVMAALVVSGAAEQGGR 87 4-20 tumor suppressor AHNK_HUMAN (Q09666) Neuroblast 5.40E-05 312292.7 Q09666 GEIDASVPELEG 88 1550-1571 differentiation-associated protein DLRGPQVDVK AHNAK (Desmoyokin) (Fragments) TTLL7_HUMAN (Q6ZT98) Tubulin tyrosine 1.22E-03 102933.8 Q6ZT98 YLLPGSTQFFLRTPTYNLK 89 849-867 ligase-like protein 7 (Protein NYD-SP30) SYCP2_HUMAN (Q9BX26) Synaptonemal 8.81E-03 175528.1 Q9BX26 ESKKLLTIILK 90 347-357 complex protein 2 (SCP-2 protein) (Synaptonemal complex lateral e DDEF2_HUMAN (O43150) Development 4.59E-03 111580.7 O43150 EIISEVQR 91 422-429 and differentiation-enhancing factor 2 (Pyk2 C-terminus associated AT8B3_HUMAN (O60423) Probable 4.37E-03 147935.6 O60423 QALMVTHKELATIK 92 282-295 phospholipid-transporting ATPase IK (EC 3.6.3.1) (ATPase class I type ST65G_HUMAN (O94864) STAGA 3.26E-03 46163.76 O94864 YWGEIPISSSQTN 93 6-30 complex 65 gamma subunit RSSFDLLPREFR (STAF65gamma) (SPTF-associated factor 65 gamma) ICT1_HUMAN (Q14197) Immature colon 1.84E-04 23615.4 Q14197 FHLATAEWIAEPVRQKIAITHK 94 103-124 carcinoma transcript 1 protein precursor (Digestion substraction FATH_HUMAN (Q14517) Cadherin-related 6.63E-03 505962.8 Q14517 EVHSEIIQVEATDK 95 836-849 tumor suppressor homolog precursor (Fat protein homolog) Q59FQ9 (Q59FQ9) DDX19-like protein 1.07E-03 35593.43 Q59FQ9 AGFAFEIPMKITWVSTVERGQK 96 21-42 variant (Fragment) HHCM_HUMAN (Q05877) Hepatocellular 4.42E-04 52117.47 Q05877 VIIISILQQVMANTLEINGK 97 390-409 carcinoma protein HHCM (HHC(M)) Q5VW08 (Q5VW08) 2.59E-03 116866.8 Q5VW08 FELQDSGSSLLPKEIVKVEK 98 362-381 OTTHUMP00000018324 (Hypothetical protein KIAA0564) Q8N8D3 (Q8N8D3) Hypothetical protein 9.18E-03 53760.11 Q8N8D3 ELKALEEALRASQEK 99 87-101 FLJ39642 Q8WZ24 (Q8WZ24) Hypothetical protein 2.81E-03 32096.25 Q8WZ24 GCWGLSCQLLEHAVRLCR 100 270-287 EVPL_HUMAN (Q92817) Envoplakin (210 kDa 9.32E-03 231475.4 Q92817 SLLEEER 101 1157-1163 paraneoplastic pemphigus antigen) (p210) (210 kDa cornified Q96RG5 (Q96RG5) Insulin receptor 9.03E-03 137347.9 Q96RG5 LEYYESEKKWR 102 75-85 substrate 2 insertion mutant (Fragment) PCDGM_HUMAN (Q9Y5F6) Protocadherin 9.32E-03 101858 Q9Y5F6 SNTLRER 103 812-818 gamma C5 precursor (PCDH-gamma-C5) Q4VXF3 (Q4VXF3) Transcription 9.75E-03 100648 Q4VXF3 QLQEFIPNIKDR 104 497-508 termination factor, RNA polymerase I (Fragment) FBXL4_HUMAN (Q9UKA2) F-box/LRR- 7.48E-03 70051.23 Q9UKA2 WEILWSERPTK 105 172-182 repeat protein 4 (F-box and leucine-rich repeat protein 4) (F-box pr IGF1A_HUMAN (P01343) Insulin-like 5.10E-03 17014.34 P01343 APQTGIVDECCFR 106 86-98 growth factor IA precursor (IGF-IA) (Somatomedin C) (Mechano grow KV1R_HUMAN (P01610) Ig kappa chain 2.86E-06 11832.84 P01610 RLIYGATSLQSGVPSR 107 46-61 V-I region WEA ANGI_HUMAN (P03950) Angiogenin 2.32E-04 16539.44 P03950 NVVVACENGLPVHLDQSIFR 108 126-145 precursor (EC 3.1.27.--) (Ribonuclease 5) (RNase 5) PLAK_HUMAN (P14923) Junction 6.43E-04 81446.77 P14923 LNTIPLFVQLLYSSVENIQR 109 581-600 plakoglobin (Desmoplakin III) AKT2_HUMAN (P31751) RAC-beta 6.72E-04 55733.18 P31751 DIKLENLMLDKDGHIK 110 275-290 serine/threonine-protein kinase (EC 2.7.1.37) (RAC-PK-beta) (Protein k

K1C20_HUMAN (P35900) Keratin, type I 7.26E-03 48456.98 P35900 VFDDLTLHKTDLEIQIEELNK 111 179-199 cytoskeletal 20 (Cytokeratin-20) (CK-20) (Keratin-20) (K20) (P FAL39_HUMAN (P49913) Antibacterial 2.44E-03 19289.16 P49913 SSDANLYR 112 50-57 protein FALL-39 precursor (FALL-39 peptide antibiotic) (Cationic PRELP_HUMAN (P51888) Prolargin 3.74E-03 43782.22 P51888 ISSVPAINNR 113 303-312 precursor (Proline-arginine-rich end leucine-rich repeat protein) CUL4A_HUMAN (Q13619) Cullin-4A (CUL- 8.78E-03 76771.75 Q13619 PLIACVEKQLLGEHLTAILQK 114 185-205 4A) DYH5_HUMAN (Q8TE73) Ciliary dynein 8.02E-04 528667.8 Q8TE73 ESRNELQITSLNHK 115 3618-3631 heavy chain 5 (Axonemal beta dynein heavy chain 5) (HL1) NRX2A_HUMAN (Q9P2S2) Neurexin-2- 4.70E-03 184864.2 Q9P2S2 SLQLSVDNVTVEGQMAGAHMR 116 834-854 alpha precursor (Neurexin II-alpha) ST1C1_HUMAN (O00338) 9.77E-03 34857.38 O00338 SILDQSISSFMR 117 247-258 Sulfotransferase 1C1 (EC 2.8.2.--) (SULT1C#1) (ST1C2) (humSULTC2) DHCA_HUMAN (P16152) Carbonyl 1.73E-03 30224.83 P16152 IGVTVLSRIHAR 118 198-209 reductase [NADPH] 1 (EC 1.1.1.184) (NADPH-dependent carbonyl reductase RM28_HUMAN (Q13084) 39S ribosomal 3.58E-03 33841.77 Q13084 EFYSEILDKKFTVTVTMR 119 144-161 protein L28, mitochondrial precursor (L28mt) (MRP-L28) (Melanoma UBR1_HUMAN (Q8IWV7) Ubiquitin-protein 1.66E-03 200079.2 Q8IWV7 TVVQSCGHSLETK 120 584-596 ligase E3 component N-recognin-1 (EC 6.--. --.--) (Ubiquitin-prot SMYD1_HUMAN (Q8NB12) SET and 8.58E-03 56579.95 Q8NB12 LKDDLFLGVKDNPK 121 283-296 MYND domain containing protein 1 NOX1_HUMAN (Q9Y5S8) NADPH oxidase 4.35E-03 64829.09 Q9Y5S8 QATDGSLASILSSLSHDEKK 122 131-150 homolog 1 (NOX-1) (NOH-1) (NADH/NADPH mitogenic oxidase subunit P O75229 (O75229) R31449_3 (Fragment) 6.40E-03 93393.15 O75229 FNIFYPDLIDK 123 556-566 Q6ZTY8 (Q6ZTY8) Hypothetical protein 2.80E-03 135743.9 Q6ZTY8 LTLARSLVLLDDLTKAEK 124 902-919 FLJ44112 TRPC4_HUMAN (Q9UBN4) Short transient 1.61E-04 112030.3 Q9UBN4 VCPFKSEKVVVEDTVPIIPK 125 927-946 receptor potential channel 4 (TrpC4) (trp-related protein 4) ( BPAEA_HUMAN (O94833) Bullous 6.56E-03 590630.3 O94833 QFHEAWSKLMEWLEESEK 126 4296-4313 pemphigoid antigen 1, isoforms 6/9/10 (Trabeculin-beta) (Bullous pemph K1C17_HUMAN (Q04695) Keratin, type I 1.37E-09 47945.07 Q04695 YCVQLSQIQGLI 127 334-356 cytoskeletal 17 (Cytokeratin-17) GSVEEQLAQLR (CK-17) (Keratin-17) (K17) (3 Q59FP4 (Q59FP4) Integrin-linked kinase 8.99E-04 13981.02 Q59FP4 TSVQQTLPLRHPLP 128 8-31 variant (Fragment) TLTRLYLASR Q6XQN6 (Q6XQN6) Nicotinate 3.83E-03 60239.51 Q6XQN6 LDSGDLLQQAQEIR 129 319-332 phosphoribosyltransferase-like protein Q6ZTY7 (Q6ZTY7) Hypothetical protein 5.85E-03 57512.96 Q6ZTY7 MARIILQDEDVTTKIDNDWK 130 177-196 FLJ44113 CM35H_HUMAN (Q9UGN4) CMRF35-H 6.75E-03 33151.84 Q9UGN4 EVEVEYSTVASPR 131 250-262 antigen precursor (CMRF35-H9) (CMRF- 35-H9) (Inhibitory receptor prote

[0041]In addition, the tandem mass spectra were analyzed using more stringent filtering criteria, with a goal of reducing false positives. In particular, the spectra were analyzed using the filtering algorithms of the Scalfold Software (Proteome Software Inc., Portland, Oreg.).

[0042]The results are provided in Table 2. In short, 86 peptides were identified as biomarkers that correlate to the disease state. Thus, evaluating patient samples for the presence of one or more of these biomarkers will provide a useful method for detecting breast cancer.

TABLE-US-00002 TABLE 2 Protein Calculated Protein molecular SEQ Peptide accession weight ID Mass Residue Protein name numbers (Da) Peptide sequence NOs (AMU) number Fibrinogen gamma chain FIBG_HUMAN 51495.3 ASTPNGYDNGIIWATWK 132 1893.914 383-399 precursor Complement factor B precursor CFAB_HUMAN 85515.2 DFHINLFQVLPWLK 133 1769.975 740-753 (EC 3.4.21.47) (C3/C5 convertase) (Properdin factor B) (Glycine-rich beta glycoprotein) (GBG) (PBF2) [Contains: Complement factor B Ba fragment; Complement factor B Bb fragment] Beta-2-glycoprotein I precursor APOH_HUMAN 38280.5 TCPKPDDLPFSTVVPLK 134 1914.005 22-38 (Apolipoprotein H) (Apo-H) (B2GPI) (Beta(2)GPI) (Activated protein C-binding protein) (APC inhibitor) (Anticardiolipin cofactor) Alpha-fetoprotein precursor FETA_HUMAN 68660.2 AENAVECFQTK 135 1296.59 195-205 (Alpha-fetoglobulin) (Alpha-1- fetoprotein) Hypothetical protein Q6MZQ6 52024.1 LSCAASGFTFR 136 1216.579 39-49 DKFZp686G11190 Keratin 10 Q14664 57231.3 GSSGGGCFGGSSGG 137 2342.985 53-79 YGGLGGFGGGSFR Apolipoprotein M (Apo-M) (ApoM) APOM_HUMAN 21235.9 KWIYHLTEGSTDLR 138 1718.887 99-112 (G3a protein) Gelsolin precursor (Actin- GELS_HUMAN 85679.8 EVQGFESATFLGYFK 139 1722.838 148-162 depolymerizing factor) (ADF) (Brevin) (AGEL) Immunoglobulin J chain IGJ_HUMAN 15576.5 SSEDPNEDIVER 140 1389.614 25-36 Kininogen-1 precursor (Alpha-2- KNG1_HUMAN 71927.5 YNSQNQSNNQFVLYR 141 1874.878 44-58 thiol proteinase inhibitor) [Contains: Kininogen-1 heavy chain; Bradykinin (Kallidin I); Lysyl-bradykinin (Kallidin II); Kininogen-1 light chain; Low molecular weight growth promoting factor] GMP synthase [glutamine- GUAA_HUMAN 76698.7 LYGAQFHPEVGLTENGK 142 1859.929 184-200 hydrolyzing] (EC 6.3.5.2) (Glutamine amidotransferase) (GMP synthetase) 1-phosphatidylinositol-4,5- PLCB3_HUMAN 138784.7 LVAGQQQVLQQLAEEEPK 143 2008.072 1150-1167 bisphosphate phosphodiesterase beta 3 (EC 3.1.4.11) (Phosphoinositide phospholipase C) (PLC-beta-3) (Phospholipase C-beta-3) Hypothetical protein FLJ45653 Q6ZSB9 85057.6 QAPDTSDGSCTELPFK 144 1752.775 193-208 Zinc finger protein 157 (HZF22) ZN157_HUMAN 58272 IQTLDQNVEYNGCR 145 1709.792 127-140 Biogenesis of lysosome-related BL1S1_HUMAN 14293.3 KELQEKR 146 930.5373 17-23 organelles complex-1, subunit 1 (BLOC-1 subunit 1) (GCN5-like protein 1) (RT14 protein) Fibrinogen beta chain precursor FIBB_HUMAN 55910.6 REEAPSLRPAP 147 2107.105 53-72 [Contains: Fibrinopeptide B] PPISGGGYR Insulin-like growth factor binding Q6P1M6 31656.1 FLNVLSPR 148 945.5522 226-233 protein 3 C18orf34 protein Q5BJE1 100177.3 LTEDNKKLEIDINK 149 1672.913 449-462 Zinc finger and BTB domain ZBTB1_HUMAN 81998.7 MDLEENPDEQSEIR 150 1704.739 488-501 containing protein 1 Nuclear factor erythroid 2 related NF2L2_HUMAN 67809.4 EKGENDK 151 819.3849 542-548 factor 2 (NF-E2 related factor 2) (NFE2-related factor 2) (Nuclear factor, erythroid derived 2, like 2) (HEBP1) Receptor interacting protein Q5RKT0 100004.6 QNLRETQKFFR 152 1466.787 43-53 kinase 5, isoform 2 Nipped-B-like protein (Delangin) NIPBL_HUMAN 316037.3 ELPPELLAEIESTMPLCER 153 2227.099 1050-1068 (SCC2 homolog) Thrombospondin-2 precursor TSP2_HUMAN 129934.9 MVWRLVLLALWVW 154 2734.466 1-23 PSTQAGHQDK Alpha-1-acid glycoprotein 2 A1AG2_HUMAN 23585.2 EQLGEFYEALDCLCIPR 155 2112.974 154-170 precursor (AGP 2) (Orosomucoid- 2) (OMD 2) Apolipoprotein B-100 precursor APOB_HUMAN 515554.3 VELEVPQLCSFILK 156 1674.914 80-93 (Apo B-100) [Contains: Apolipoprotein B-48 (Apo B-48)] Hypothetical protein FLJ44261 Q6ZTS6 109100.2 APEPDLMSPTR 157 1213.589 821-831 CMRF35-H antigen precursor CM35H_HUMAN 33154.7 EVEVEYSTVASPR 158 1465.717 250-262 (CMRF35-H9) (CMRF-35-H9) (Inhibitory receptor protein 60) (IRp60) (IRC1/IRC2) (NK inhibitory receptor) Two-pore calcium channel protein Q8NHX9 85283.9 SYGSVLLSAEEFQK 159 1557.78 385-398 2 (Two pore segment channel 2) Protocadherin beta 14 precursor PCDBE_HUMAN 87532.2 DINDHSPTFLDK 160 1401.665 125-136 (PCDH-beta14) Dimethylglycine dehydrogenase, M2GD_HUMAN 96790.9 LNKPADFIGK 161 1102.626 748-757 mitochondrial precursor (EC 1.5.99.2) (ME2GLYDH) Angiopoietin-1 receptor precursor TIE2_HUMAN 125795 FNPICKASGWP 162 2532.263 366-387 (EC 2.7.1.112) (Tyrosine-protein LPTNEEMTLVK kinase receptor TIE-2) (Tyrosine- protein kinase receptor TEK) (P140 TEK) (Tunica interna endothelial cell kinase) (CD202b antigen) MGC5297 protein (Fragment) Q9BVD3 62640.1 VLTPYCYTIDVEIK 163 1713.878 446-459 Dystrobrevin beta (Beta- DTNB_HUMAN 71338.1 LIARYAARLAAEAGNVTR 164 1916.083 404-421 dystrobrevin) (DTN-B) A-kinase anchor protein 3 AKAP3_HUMAN 94719.5 SCDASLAELGDDKSGDASR 165 1953.846 687-705 (Protein kinase A anchoring protein 3) (PRKA3) (A-kinase anchor protein 110 kDa) (AKAP 110) (Sperm oocyte binding protein) (Fibrousheathin I) (Fibrous sheath protein of 95 kDa) (FSP95) Netrin receptor UNC5C precursor UNC5C_HUMAN 103084.7 VYEMYVTVHR 166 1296.641 564-573 (Unc-5 homolog C) (Unc-5 homolog 3) EPM2A-interacting protein 1 EPMIP_HUMAN 70352.8 ILSIDRNLRNQLFNR 167 1872.057 154-168 (Laforin-interacting protein) Transmembrane protein 2 Q9UHN6 154359 YVGTGGIDQK 168 1037.527 847-856 Symplekin SYMPK_HUMAN 141135.5 NMPSSKDTRK 169 1163.584 338-347 Hook homolog 3 (hHK3) HOOK3_HUMAN 83110 RAIIEDLEPR 170 1211.675 566-575 Hypothetical protein SERAC1 Q6PKF3 74145.6 KDAFLYQRTLQFIR 171 1798.997 632-645 39S ribosomal protein L28, RM28_HUMAN 33846.3 EFYSEILDKKFTVTVTMR 172 2207.142 144-161 mitochondrial precursor (L28mt) (MRP-L28) (Melanoma antigen p15) (Melanoma-associated antigen recognized by T lymphocytes) Seven transmembrane helix Q8NGB0 156486.6 WLSSPVFSLRR 173 1347.754 334-344 receptor Lymphotactin precursor (XCL1) XCL1_HUMAN 12498.7 AVIFITKRGLK 174 1245.805 57-67 (Cytokine SCM-1) (ATAC) (Lymphotaxin) (SCM-1-alpha) (Small inducible cytokine C1) (XC chemokine ligand 1) Peptidoglycan recognition protein PGRP_HUMAN 21712.6 VPTPQAIRAAQGLLA 175 3122.726 139-168 precursor (PGRP-S) CGVAQGALRSNYVLK Ankyrin repeat domain protein ANR18_HUMAN 115652.2 NQANIHAVDNFK 176 1370.682 189-200 18A Mitochondrial ribosomal protein Q9BYD0 28446.1 VLSPYDLTHK 177 1172.632 229-238 L16 (L16mt) Protein disulfide-isomerase A4 PDIA4_HUMAN 72916 NNKGPVKVVVGK 178 1238.759 522-533 precursor (EC 5.3.4.1) (Protein ERp-72) (ERp72) 2-5A-dependent ribonuclease RN5A_HUMAN 83516.7 RGANVNLR 179 899.5175 147-154 (EC 3.1.26.--) (2-5A-dependent RNase) (Ribonuclease L) (RNase L) (Ribonuclease 4) WD-repeat protein 75 WDR75_HUMAN 94483 SEQPTLVTASKDGYFK 180 1770.892 456-471 Zinc finger protein 294 ZN294_HUMAN 200525.3 VFKMLLGDEKQSIVQAK 181 1934.079 629-645 Hypothetical protein FLJ44356 Q6ZTQ6 26752.7 GADPPPPPSRTGR 182 1304.671 59-71 Protein C6orf149 CF149_HUMAN 10740.9 ENKNVKDPVEIQTLVNK 183 1968.077 42-58

Ubiquitin specific protease 48 Q5SZI4 18677.1 FNDEDIEKMEGKK 184 1582.743 47-59 Intercellular adhesion molecule 3 ICAM3_HUMAN 59363.8 ADQEGAREIVC 185 2230.1 284-303 precursor (ICAM-3) (ICAM-R) NVTLGGERR (CDw50) (CD50 antigen) Hypothetical protein Q86VG6 11513.3 AGECVTAGGLGGARRR 186 1587.814 12-27 Zinc finger protein HRX (ALL-1) HRX_HUMAN 431732.3 QVSQPALVIPP 187 2080.156 1297-1316 (Trithorax-like protein) QPPTTGPPR Hypothetical protein FLJ37300 Q52M87 61958.1 HLLTIAGWKHEK 188 1432.807 457-468 Insulin-like growth factor IA IGF1A_HUMAN 17008.1 APQTGIVDECCFR 189 1552.689 86-98 precursor (IGF-IA) (Somatomedin C) (Mechano growth factor) (MGF) Guanine nucleotide exchange MCF2L_HUMAN 123968.6 FKPMQRHLFLHEK 190 1710.927 870-882 factor DBS (DBL's big sister) (MCF2 transforming sequence- like protein) (Fragment) Hypothetical protein FLJ40235 Q8N7X8 21265.9 VRASQELEMSLK 191 1390.736 160-171 Novel protein Q9H4G2 164839.6 EKLSLEPVLPARNPNR 192 1833.035 309-324 Chromosome 9 open reading Q5SZB4 47622.5 EDPDFLGAFLGELLPSR 193 1875.95 134-150 frame 50 Piccolo protein (Aczonin) PCLO_HUMAN 566639.4 VMSDGPVKPEGAK 194 1314.673 4843-4855 Hypothetical protein FLJ90556 Q8N2J3 30614.7 DSGGQTSAGCPSGWLGTR 195 1793.788 123-140 Prokineticin receptor 2 (PK-R2) PKR2_HUMAN 43979 YLAIVHPLKPR 196 1306.8 154-164 (G-protein coupled receptor 73- like 1) (GPR73b) (GPRg2) 1-phosphatidylinositol-4,5- PLCB2_HUMAN 133665.8 QAACLEQIREMEK 197 1605.773 1101-1113 bisphosphate phosphodiesterase beta 2 (EC 3.1.4.11) (Phosphoinositide phospholipase C) (PLC-beta-2) (Phospholipase C-beta-2) Reticulon-4 (Neurite outgrowth RTN4_HUMAN 129916.6 LSALPPEGGKPYLE 198 2604.393 788-811 inhibitor) (Nogo protein) (Foocen) SFKLSLDNTK (Neuroendocrine-specific protein) (NSP) (Neuroendocrine-specific protein C homolog) (RTN-x) (Reticulon-5) Tripartite motif protein 8 (RING TRIM8_HUMAN 61469.3 QTVEVLDK 199 931.5102 249-256 finger protein 27) (Glioblastoma- expressed RING finger protein) Beta-adducin (Erythrocyte ADDB_HUMAN 80836.4 TTWMKADEVEK 200 1337.641 460-470 adducin beta subunit) Hypothetical protein FLJ13755 Q9H8C8 148979 FDLKQWLSATK 201 1336.727 601-611 Elastin precursor (Tropoelastin) ELN_HUMAN 68478.6 FPGVGVLPGVPTGAGVK 202 1551.89 160-176 Hypothetical protein PRSS35 Q8N3Z0 47052.9 YAQICLWIHGNDANCAYG 203 2125.922 396-413 (ENML522) Diacylglycerol kinase, beta (EC DGKB_HUMAN 90578.9 CLRWGGGYEGENLMK 204 1769.811 532-546 2.7.1.107) (Diglyceride kinase) (DGK-beta) (DAG kinase beta) (90 kDa diacylglycerol kinase) Hypothetical protein Q6PJ41 3537.6 MIMILVLLSLGR 205 1358.827 1-12 Zinc finger protein 592 ZN592_HUMAN 137535.7 GSDLPPDPHNCGK 206 1393.617 97-109 Envoplakin (210 kDa EVPL_HUMAN 231600.4 SLLEEER 207 875.4474 1157-1163 paraneoplastic pemphigus antigen) (p210) (210 kDa cornified envelope precursor protein) ATP-binding cassette sub-family ABCA2_HUMAN 269960.8 ARRFLWNLILDLIK 208 1771.075 2221-2234 A member 2 (ATP-binding cassette transporter 2) (ATP- binding cassette 2) Putative helicase Mov10l1 (EC M10L1_HUMAN 135276.7 RFNVAITR 209 976.5691 1131-1138 3.6.1.--) (Moloney leukemia virus 10-like protein 1) (MOV10-like 1) Novel protein (MGC26989) Q5T1M9 62944.4 PLAIAKQASFSSK 210 1347.764 279-291 Tic O95621 116328.1 SMSCQEFITNLNGLR 211 1769.832 702-716 Hypothetical protein FLJ46306 Q6ZRJ5 18924.6 EKEQEAGGVSYWDIGK 212 1795.851 35-50 Hypothetical class II basic helix- Q7RTU2 32314.4 RQRGDAGSPWGCPLCPDR 213 2084.951 151-168 loop-helix protein MESP2 (Fragment) RSK-like protein (Ribosomal Q8TDD3 118668.4 RNPEDVQEIIVWKR 214 1781.966 40-53 protein S6 kinase, 52 kDa, polypeptide 1) Histone H2B-related protein (H2B Q7Z2G1 19581.4 LAESEGTK 215 834.421 153-160 histone family, member W, testis- specific) LOC493860 protein Q6P5Q7 45467 FLNLQNEHEKALGTWKR 216 2084.104 237-253 Glutamine synthetase (EC GLNA_HUMAN 41915.7 TCLLNETGDEPFQYK 217 1814.827 357-371 6.3.1.2) (Glutamate--ammonia ligase) (GS)

Example 2

Cancer-Related Peptides are not Necessarily Biomarkers

[0043]The above methods showed that a number of peptides previously known to be associated with breast cancer were not indicative of a disease state, and, thus, not useful as a biomarker. Examples include, RUN and FYVE domain-containing 1 variant (fragment), haptoglobin precursor that contains: haptoglobin alpha chain and haptoglobin beta chain, tetranectin precursor (TN) (Plasminogen-kringle 4 binding protein), vitamin D-binding protein, apolipoprotein C-IV precursor (Apo-CIV or ApoC-IV), VH1 protein precursor (fragment), Ig kappa chain V-III region SIE, hypothetical protein Q569I7, haptoglobin-related protein precursor, breast carcinoma amplified sequence 1 (novel amplified in breast cancer 1) (amplified and overexpressed in breast cancer), sodium-D-glucose cotransporter (regulatory solute carrier protein, family 1, member 1), complement component C8 gamma chain precursor, Ig heavy chain V-III region TIL, hypothetical protein DKFZp686I04196 (fragment), alpha-2-macroglobulin precursor (Alpha-2-M), hypothetical protein DKFZp761P18121, fibrinogen alpha chain precursor that contains fibrinopeptide A, IGHG1 protein, hypothetical protein MGC27016, seprase (EC 3.4.21.-) (fibroblast activation protein alpha) (integral membrane serine protease) (170-kDa melanoma membrane-bound gelatinase), PTPL1-associated RhoGAP, zinc finger protein 385 (hematopoietic zinc finger protein) (retinal zinc finger protein), hypothetical protein FLJ45950, hypothetical protein FLJ39462, smoothelin, latrophilin-3 precursor (calcium-independent alpha-latrotoxin receptor 3) (lectomedin-3), type III iodothyronine deiodinase (EC 1.97.1.11) (type-III 5'deiodinase) (DIOIII) (type 3 DI) (5DIII).

Sequence CWU 1

217113PRTHomo sapiens 1Gln Trp Thr Gln Gln His Leu Asp Ala Ala Asp Leu Arg1 5 10215PRTHomo sapiens 2Gln Ala Ser Thr Leu Ala Asp Leu Asp Ser Gly Asn Met Glu Lys1 5 10 15317PRTHomo sapiens 3Asn Leu Arg Phe Glu Ile Asn Cys Ile Pro Asn Leu Ile Glu Tyr Val1 5 10 15Lys419PRTHomo sapiens 4Lys Glu Val Gly Asp Val Ser Ile Leu Ile Asn Asn Ala Gly Ile Val1 5 10 15Thr Gly Lys517PRTHomo sapiens 5Asn Asn Asn Thr Asp Leu Met Ile Leu Lys Asn Thr Lys Asp Ala Val1 5 10 15Arg617PRTHomo sapiens 6Gly Ser Ile Glu Ile Leu Arg Asp Asp Val Val Val Ala Ile Leu Gly1 5 10 15Lys724PRTHomo sapiens 7Thr Ala Ser Ala Leu Phe Ala Gly Phe Arg Ala Leu Gly Leu Phe Ser1 5 10 15Asn Asp Ile Pro His Val Val Arg 20810PRTHomo sapiens 8Glu Arg Val Thr Pro Pro Ile Ser Glu Arg1 5 10914PRTHomo sapiens 9Ala Ala Gly Ser Asp Ala Asp His Leu Arg Glu Gln Gln Arg1 5 101017PRTHomo sapiens 10Pro Asn Lys Val Asn Phe Gly Thr Asp Phe Leu Thr Ala Ile Lys Asn1 5 10 15Arg1113PRTHomo sapiens 11Thr Pro Lys Cys Thr Val Leu Leu Ser Leu Ile Gln Arg1 5 101211PRTHomo sapiens 12Asp Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg1 5 101317PRTHomo sapiens 13Thr Leu Lys Leu Lys Tyr Ala Glu Leu Ala Ala Leu Glu Phe Pro Pro1 5 10 15Lys1414PRTHomo sapiens 14Ser Pro Ala Gln Ala Glu Leu Ser Tyr Leu Asn Lys Ala Lys1 5 101511PRTHomo sapiens 15Arg Leu Gly Val Thr Leu Val Gly His Gln Lys1 5 101618PRTHomo sapiens 16Glu Val Glu Leu Tyr Val Thr Glu Ile Glu Lys Glu Lys Glu Glu Ala1 5 10 15Glu Lys1721PRTHomo sapiens 17Asp Ser Leu Thr Leu His Thr Lys Pro Glu Pro Leu Glu Gly Pro Ala1 5 10 15Leu Ser His Ser Val 201825PRTHomo sapiens 18Glu Asp Val Asp Pro His Asn Gly Ala Asp Asp Val Phe Ser Ser Ser1 5 10 15Gly Ser Leu Gly Lys Ala Ser Glu Lys 20 251916PRTHomo sapiens 19Glu Val Gly Asn Leu Leu Leu Glu Asn Ser Gln Leu Leu Glu Thr Lys1 5 10 152013PRTHomo sapiens 20Ser Asp Leu Ala Val Pro Ser Glu Leu Ala Leu Leu Lys1 5 102121PRTHomo sapiens 21Ser Pro Pro Leu Leu Glu Ser Pro Asp Ala Thr Arg Glu Ser Met Val1 5 10 15Lys Leu Ser Ser Lys 202217PRTHomo sapiens 22Glu Ala Cys Ile Val Glu Ala Leu Gly Ile Gln Thr Leu Thr Asn Gln1 5 10 15Lys2314PRTHomo sapiens 23Met Ala Ala Gly Glu Pro Arg Ser Leu Leu Phe Phe Gln Lys1 5 102414PRTHomo sapiens 24Met Val Phe His Ile Thr Thr Gly Ser Gln Glu Phe Asp Lys1 5 102516PRTHomo sapiens 25Phe Ile Gly Ser Leu His Ser Tyr Ser Phe Ser Ser Lys His Thr Arg1 5 10 152624PRTHomo sapiens 26Pro Ala Val Trp Leu Ser Trp Lys Val Ser Gly Pro Ala Ala Pro Ala1 5 10 15Gln Ser Tyr Thr Ala Leu Phe Arg 202713PRTHomo sapiens 27Tyr Ile Gly Ile Arg Met Met Ser Leu Thr Ser Ser Lys1 5 102816PRTHomo sapiens 28Asn Leu Tyr Leu Ala Trp Asn Cys Tyr Phe Asn Lys Val Cys Glu Lys1 5 10 152915PRTHomo sapiens 29Val Glu Met Ala Asn Leu Leu Asn Leu Thr Glu Arg Gln Ile Lys1 5 10 153030PRTHomo sapiens 30Leu Val Glu Val Ile Pro Glu Gly Ala Met Leu Arg Leu Gly Met Thr1 5 10 15Asn Pro Pro Tyr Ile Leu Glu His Leu Glu Glu Met Ala Lys 20 25 303124PRTHomo sapiens 31Pro Ala Ala Ser Pro Asn Thr Thr Ser Ser Arg Gly Gln Thr Val His1 5 10 15Pro Pro Cys Ser Ser Lys Leu Arg 203221PRTHomo sapiens 32Asn Arg Leu Thr Asp Tyr Tyr Glu Pro Leu Leu Lys Asn Asn Ser Thr1 5 10 15Ala Tyr Ser Thr Arg 203315PRTHomo sapiens 33Phe Ala Met Pro Ser Ser Thr Pro Leu Tyr His Asp Ala Ile Lys1 5 10 153419PRTHomo sapiens 34Ser Cys Asp Ala Ser Leu Ala Glu Leu Gly Asp Asp Lys Ser Gly Asp1 5 10 15Ala Ser Arg359PRTHomo sapiens 35Val Thr Ile Ser Val Asp Thr Ser Lys1 53614PRTHomo sapiens 36Met Asp Ile Asn Thr Tyr Asn Asn Gln Leu His Leu Gln Arg1 5 103711PRTHomo sapiens 37Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys1 5 103824PRTHomo sapiens 38Ser His Leu Thr Val Cys Phe Leu Pro Ser Val Pro Phe Leu Ile Leu1 5 10 15Val Ser Thr Leu Ala Thr Ala Lys 203919PRTHomo sapiens 39Glu Leu Ser Glu Asn Ile Asn Ala Val Thr Leu Met Lys Glu Glu Leu1 5 10 15Lys Glu Lys4023PRTHomo sapiens 40Pro Gly Gln Asn Ser Cys Arg Asp Ser Asp Ser Glu Ser Ala Ser Gly1 5 10 15Glu Ser Lys Gly Phe His Arg 204116PRTHomo sapiens 41Asp Ala Asn Ser Val Ile Thr Tyr Gln Leu Thr Gly Gly Asn Thr Arg1 5 10 154222PRTHomo sapiens 42Asp Glu Ile Gln Cys Val Ile Ala Thr Ile Ala Phe Gly Met Gly Ile1 5 10 15Asn Lys Ala Asp Ile Arg 204324PRTHomo sapiens 43Ala Val Ile Lys Asn Arg Asn Thr Thr Tyr Ile Val Ile Gly Ala Val1 5 10 15Gln Pro Asn Asp Ile Ser Asn Lys 204427PRTHomo sapiens 44Val Met Gln Leu Gln Tyr Glu Asn Arg Val Leu Met Ser Asn Met Gln1 5 10 15Arg Tyr Asp Leu Ala Ser His Leu Gly Ile Arg 20 254521PRTHomo sapiens 45Gly Arg Ile Leu Ser Gly Asn Phe Phe Leu Leu Lys Glu Asn Tyr Glu1 5 10 15Asn Tyr Phe Val Lys 204610PRTHomo sapiens 46Asp Arg Asp Gly Arg Pro Gln Val Val Arg1 5 104715PRTHomo sapiens 47Asn Val Glu Val Thr Val Ser Val Tyr Asp Glu Asp Gly Lys Arg1 5 10 154822PRTHomo sapiens 48Lys Gly Gln Glu Gly Glu Ser Cys Leu Arg Thr Phe Asp Cys Gly Pro1 5 10 15Gly Leu Cys Cys Ala Arg 204912PRTHomo sapiens 49Thr Cys Cys His Ile Met Leu Ser Gln Glu Glu Lys1 5 105019PRTHomo sapiens 50Cys Lys Asp Gly Leu Gly Glu Tyr Thr Cys Thr Cys Leu Glu Gly Phe1 5 10 15Glu Gly Lys5111PRTHomo sapiens 51Ala Val Ile Phe Ile Thr Lys Arg Gly Leu Lys1 5 105218PRTHomo sapiens 52Gln Glu Asn Glu Lys Pro Leu Pro Glu Asn Met Asp Ala Phe Glu Lys1 5 10 15Val Arg5319PRTHomo sapiens 53Ala Lys Glu Phe Ile Ile Gly Thr Gly Trp Glu Glu Ala Val Gln Gly1 5 10 15Trp Gly Arg5416PRTHomo sapiens 54Leu Pro Lys Gly Met Gln Ala Arg Ala Pro Ser Gln Tyr Ser Thr Arg1 5 10 155515PRTHomo sapiens 55Glu Glu Ala Arg Glu Glu Phe Leu Ser Ala Ser Pro Glu Pro Arg1 5 10 155611PRTHomo sapiens 56Leu Asp Thr Ser Gln Trp Pro Leu Leu Leu Lys1 5 105719PRTHomo sapiens 57Glu Glu Ala Pro Ser Leu Arg Pro Ala Pro Pro Pro Ile Ser Gly Gly1 5 10 15Gly Tyr Arg5825PRTHomo sapiens 58Asp Trp Ser His Tyr Phe Lys Ile Ile Glu Asp Leu Arg Ala Gln Ile1 5 10 15Phe Ala Asn Thr Val Asp Asn Ala Arg 20 25598PRTHomo sapiens 59Phe Leu Asn Val Leu Ser Pro Arg1 56011PRTHomo sapiens 60Gln Asn Cys Lys Gly Asp Ser Gln Pro Asn Lys1 5 106118PRTHomo sapiens 61Val Val Thr Leu Ala Gln His Ile Ser Glu Val Ile Thr Gln Asp Tyr1 5 10 15Thr Arg6216PRTHomo sapiens 62Ile Cys Asp Gln Trp Asp Asn Leu Gly Ala Leu Thr Gln Lys Arg Arg1 5 10 156318PRTHomo sapiens 63Leu Gly Gln Ser Ser Met Leu Gly Glu Gln Gly Ala Arg Ile Gln Ser1 5 10 15Val Lys6421PRTHomo sapiens 64Ser Cys Gln Ser Gly Glu Asp Glu Glu Glu Asp Glu Asp Lys Glu Lys1 5 10 15Thr Phe Glu Glu Lys 206517PRTHomo sapiens 65Ala Leu Ser Asp Val Thr Asp Glu Glu Glu Val Gln Val Asp Pro Arg1 5 10 15Lys6617PRTHomo sapiens 66Asp Val Lys Asp Thr Val Thr Glu Ile Leu Asp Thr Lys Val Phe Ser1 5 10 15Tyr6713PRTHomo sapiens 67Cys Phe Val Glu Ser Leu Ser Ser Val Glu Thr Leu Lys1 5 106812PRTHomo sapiens 68Pro Cys Trp Glu Leu Lys Lys Ile Met Ile Leu Lys1 5 106926PRTHomo sapiens 69Asp Ile Gly Val Pro Met Thr Ser Val Arg Leu Pro Cys Tyr Phe Glu1 5 10 15Asn Leu Leu Ser His Phe Leu Pro Gln Lys 20 257020PRTHomo sapiens 70Ala Asp Ser Val Ala Asn Gln Gly Thr Lys Val Glu Gly Ile Thr Asn1 5 10 15Gln Gly Lys Lys 207111PRTHomo sapiens 71Ala Glu Asn Thr Ala Val Tyr Tyr Cys Ala Arg1 5 107222PRTHomo sapiens 72Glu Pro Leu Ile Ile His Trp Ala Ala Ser Pro Gln Arg Ile Glu Glu1 5 10 15Cys Val Leu Ser Gly Lys 207313PRTHomo sapiens 73Met Asp Val Asp Lys Glu Leu Gly Met Glu Ser Val Lys1 5 107420PRTHomo sapiens 74Asp Val Ala Gly Ala Arg Gly Ala Pro Pro Ala Trp Gly Gln Ala Pro1 5 10 15Ser Pro Arg Arg 207515PRTHomo sapiens 75Glu Thr Tyr Gly His Leu Gly Ala Leu Gly Cys Ala Gly Pro Lys1 5 10 157611PRTHomo sapiens 76Lys Lys Ile Leu Asp Ala Asp Ile Gln Leu Lys1 5 107730PRTHomo sapiens 77Pro Lys Met Leu Val Ile Ser Gly Gly Asp Gly Tyr Glu Asp Phe Arg1 5 10 15Leu Ser Ser Gly Gly Gly Ser Ser Ser Glu Thr Val Gly Arg 20 25 307811PRTHomo sapiens 78Glu Pro Glu Thr Gln Pro Val Cys Phe Phe Arg1 5 107923PRTHomo sapiens 79Gly Pro Leu Leu Ala Ala Cys Ser Ser Glu Ser Arg Glu Leu Cys Phe1 5 10 15Val Ala Leu Cys His Val Arg 208022PRTHomo sapiens 80Gly Ala Ser Ser Ser Thr Asn Asp Ala Ser Val Pro Thr Thr Lys Glu1 5 10 15Phe Glu Thr Leu Ile Lys 208114PRTHomo sapiens 81Asn Lys Pro Gly Val Tyr Thr Lys Val Tyr Asn Tyr Val Lys1 5 108225PRTHomo sapiens 82Gly Thr Leu Met Glu Leu Gly Ile Ser Pro Ile Val Thr Ser Gly Leu1 5 10 15Ile Met Gln Leu Leu Ala Gly Ala Lys 20 258314PRTHomo sapiens 83Glu Asn Lys Phe Pro Leu Ser Asn Gln Asn Met Leu Leu Arg1 5 108415PRTHomo sapiens 84Gly Arg Gly Ser Gln Gly Asn Leu Ile Phe Gly Lys Gly Thr Lys1 5 10 15858PRTHomo sapiens 85Ile Ser Ser Thr Ser Thr Asp Arg1 5868PRTHomo sapiens 86Gln Thr Val Glu Val Leu Asp Lys1 58717PRTHomo sapiens 87Gln Val Met Ala Ala Leu Val Val Ser Gly Ala Ala Glu Gln Gly Gly1 5 10 15Arg8822PRTHomo sapiens 88Gly Glu Ile Asp Ala Ser Val Pro Glu Leu Glu Gly Asp Leu Arg Gly1 5 10 15Pro Gln Val Asp Val Lys 208919PRTHomo sapiens 89Tyr Leu Leu Pro Gly Ser Thr Gln Phe Phe Leu Arg Thr Pro Thr Tyr1 5 10 15Asn Leu Lys9011PRTHomo sapiens 90Glu Ser Lys Lys Leu Leu Thr Ile Ile Leu Lys1 5 10918PRTHomo sapiens 91Glu Ile Ile Ser Glu Val Gln Arg1 59214PRTHomo sapiens 92Gln Ala Leu Met Val Thr His Lys Glu Leu Ala Thr Ile Lys1 5 109325PRTHomo sapiens 93Tyr Trp Gly Glu Ile Pro Ile Ser Ser Ser Gln Thr Asn Arg Ser Ser1 5 10 15Phe Asp Leu Leu Pro Arg Glu Phe Arg 20 259422PRTHomo sapiens 94Phe His Leu Ala Thr Ala Glu Trp Ile Ala Glu Pro Val Arg Gln Lys1 5 10 15Ile Ala Ile Thr His Lys 209514PRTHomo sapiens 95Glu Val His Ser Glu Ile Ile Gln Val Glu Ala Thr Asp Lys1 5 109622PRTHomo sapiens 96Ala Gly Phe Ala Phe Glu Ile Pro Met Lys Ile Thr Trp Val Ser Thr1 5 10 15Val Glu Arg Gly Gln Lys 209720PRTHomo sapiens 97Val Ile Ile Ile Ser Ile Leu Gln Gln Val Met Ala Asn Thr Leu Glu1 5 10 15Ile Asn Gly Lys 209820PRTHomo sapiens 98Phe Glu Leu Gln Asp Ser Gly Ser Ser Leu Leu Pro Lys Glu Ile Val1 5 10 15Lys Val Glu Lys 209915PRTHomo sapiens 99Glu Leu Lys Ala Leu Glu Glu Ala Leu Arg Ala Ser Gln Glu Lys1 5 10 1510018PRTHomo sapiens 100Gly Cys Trp Gly Leu Ser Cys Gln Leu Leu Glu His Ala Val Arg Leu1 5 10 15Cys Arg1017PRTHomo sapiens 101Ser Leu Leu Glu Glu Glu Arg1 510211PRTHomo sapiens 102Leu Glu Tyr Tyr Glu Ser Glu Lys Lys Trp Arg1 5 101037PRTHomo sapiens 103Ser Asn Thr Leu Arg Glu Arg1 510412PRTHomo sapiens 104Gln Leu Gln Glu Phe Ile Pro Asn Ile Lys Asp Arg1 5 1010511PRTHomo sapiens 105Trp Glu Ile Leu Trp Ser Glu Arg Pro Thr Lys1 5 1010613PRTHomo sapiens 106Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg1 5 1010716PRTHomo sapiens 107Arg Leu Ile Tyr Gly Ala Thr Ser Leu Gln Ser Gly Val Pro Ser Arg1 5 10 1510820PRTHomo sapiens 108Asn Val Val Val Ala Cys Glu Asn Gly Leu Pro Val His Leu Asp Gln1 5 10 15Ser Ile Phe Arg 2010920PRTHomo sapiens 109Leu Asn Thr Ile Pro Leu Phe Val Gln Leu Leu Tyr Ser Ser Val Glu1 5 10 15Asn Ile Gln Arg 2011016PRTHomo sapiens 110Asp Ile Lys Leu Glu Asn Leu Met Leu Asp Lys Asp Gly His Ile Lys1 5 10 1511121PRTHomo sapiens 111Val Phe Asp Asp Leu Thr Leu His Lys Thr Asp Leu Glu Ile Gln Ile1 5 10 15Glu Glu Leu Asn Lys 201128PRTHomo sapiens 112Ser Ser Asp Ala Asn Leu Tyr Arg1 511310PRTHomo sapiens 113Ile Ser Ser Val Pro Ala Ile Asn Asn Arg1 5 1011421PRTHomo sapiens 114Pro Leu Ile Ala Cys Val Glu Lys Gln Leu Leu Gly Glu His Leu Thr1 5 10 15Ala Ile Leu Gln Lys 2011514PRTHomo sapiens 115Glu Ser Arg Asn Glu Leu Gln Ile Thr Ser Leu Asn His Lys1 5 1011621PRTHomo sapiens 116Ser Leu Gln Leu Ser Val Asp Asn Val Thr Val Glu Gly Gln Met Ala1 5 10 15Gly Ala His Met Arg 2011712PRTHomo sapiens 117Ser Ile Leu Asp Gln Ser Ile Ser Ser Phe Met Arg1 5 1011812PRTHomo sapiens 118Ile Gly Val Thr Val Leu Ser Arg Ile His Ala Arg1 5 1011918PRTHomo sapiens 119Glu Phe Tyr Ser Glu Ile Leu Asp Lys Lys Phe Thr Val Thr Val Thr1 5 10 15Met Arg12013PRTHomo sapiens 120Thr Val Val Gln Ser Cys Gly His Ser Leu Glu Thr Lys1 5 1012114PRTHomo sapiens 121Leu Lys Asp Asp Leu Phe Leu Gly Val Lys Asp Asn Pro Lys1 5 1012220PRTHomo sapiens 122Gln Ala Thr Asp Gly Ser Leu Ala Ser Ile Leu Ser Ser Leu Ser His1 5 10 15Asp Glu Lys Lys 2012311PRTHomo sapiens 123Phe Asn Ile Phe Tyr Pro Asp Leu Ile Asp Lys1 5 1012418PRTHomo sapiens 124Leu Thr Leu Ala Arg Ser Leu Val Leu Leu Asp Asp Leu Thr Lys Ala1 5 10 15Glu Lys12520PRTHomo sapiens 125Val Cys Pro Phe Lys Ser Glu Lys Val Val Val Glu Asp Thr Val Pro1 5 10 15Ile Ile Pro Lys 2012618PRTHomo sapiens 126Gln Phe His Glu Ala Trp Ser Lys Leu Met Glu Trp Leu Glu Glu Ser1 5 10 15Glu Lys12723PRTHomo sapiens 127Tyr Cys Val Gln Leu Ser Gln Ile Gln Gly Leu Ile Gly Ser Val Glu1 5 10 15Glu Gln Leu Ala Gln Leu Arg 2012824PRTHomo sapiens 128Thr Ser Val Gln Gln Thr Leu Pro Leu Arg His

Pro Leu Pro Thr Leu1 5 10 15Thr Arg Leu Tyr Leu Ala Ser Arg 2012914PRTHomo sapiens 129Leu Asp Ser Gly Asp Leu Leu Gln Gln Ala Gln Glu Ile Arg1 5 1013020PRTHomo sapiens 130Met Ala Arg Ile Ile Leu Gln Asp Glu Asp Val Thr Thr Lys Ile Asp1 5 10 15Asn Asp Trp Lys 2013113PRTHomo sapiens 131Glu Val Glu Val Glu Tyr Ser Thr Val Ala Ser Pro Arg1 5 1013217PRTHomo sapiens 132Ala Ser Thr Pro Asn Gly Tyr Asp Asn Gly Ile Ile Trp Ala Thr Trp1 5 10 15Lys13314PRTHomo sapiens 133Asp Phe His Ile Asn Leu Phe Gln Val Leu Pro Trp Leu Lys1 5 1013417PRTHomo sapiens 134Thr Cys Pro Lys Pro Asp Asp Leu Pro Phe Ser Thr Val Val Pro Leu1 5 10 15Lys13511PRTHomo sapiens 135Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys1 5 1013611PRTHomo sapiens 136Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg1 5 1013727PRTHomo sapiens 137Gly Ser Ser Gly Gly Gly Cys Phe Gly Gly Ser Ser Gly Gly Tyr Gly1 5 10 15Gly Leu Gly Gly Phe Gly Gly Gly Ser Phe Arg 20 2513814PRTHomo sapiens 138Lys Trp Ile Tyr His Leu Thr Glu Gly Ser Thr Asp Leu Arg1 5 1013915PRTHomo sapiens 139Glu Val Gln Gly Phe Glu Ser Ala Thr Phe Leu Gly Tyr Phe Lys1 5 10 1514012PRTHomo sapiens 140Ser Ser Glu Asp Pro Asn Glu Asp Ile Val Glu Arg1 5 1014115PRTHomo sapiens 141Tyr Asn Ser Gln Asn Gln Ser Asn Asn Gln Phe Val Leu Tyr Arg1 5 10 1514217PRTHomo sapiens 142Leu Tyr Gly Ala Gln Phe His Pro Glu Val Gly Leu Thr Glu Asn Gly1 5 10 15Lys14318PRTHomo sapiens 143Leu Val Ala Gly Gln Gln Gln Val Leu Gln Gln Leu Ala Glu Glu Glu1 5 10 15Pro Lys14416PRTHomo sapiens 144Gln Ala Pro Asp Thr Ser Asp Gly Ser Cys Thr Glu Leu Pro Phe Lys1 5 10 1514514PRTHomo sapiens 145Ile Gln Thr Leu Asp Gln Asn Val Glu Tyr Asn Gly Cys Arg1 5 101467PRTHomo sapiens 146Lys Glu Leu Gln Glu Lys Arg1 514720PRTHomo sapiens 147Arg Glu Glu Ala Pro Ser Leu Arg Pro Ala Pro Pro Pro Ile Ser Gly1 5 10 15Gly Gly Tyr Arg 201488PRTHomo sapiens 148Phe Leu Asn Val Leu Ser Pro Arg1 514914PRTHomo sapiens 149Leu Thr Glu Asp Asn Lys Lys Leu Glu Ile Asp Ile Asn Lys1 5 1015014PRTHomo sapiens 150Met Asp Leu Glu Glu Asn Pro Asp Glu Gln Ser Glu Ile Arg1 5 101517PRTHomo sapiens 151Glu Lys Gly Glu Asn Asp Lys1 515211PRTHomo sapiens 152Gln Asn Leu Arg Glu Thr Gln Lys Phe Phe Arg1 5 1015319PRTHomo sapiens 153Glu Leu Pro Pro Glu Leu Leu Ala Glu Ile Glu Ser Thr Met Pro Leu1 5 10 15Cys Glu Arg15423PRTHomo sapiens 154Met Val Trp Arg Leu Val Leu Leu Ala Leu Trp Val Trp Pro Ser Thr1 5 10 15Gln Ala Gly His Gln Asp Lys 2015517PRTHomo sapiens 155Glu Gln Leu Gly Glu Phe Tyr Glu Ala Leu Asp Cys Leu Cys Ile Pro1 5 10 15Arg15614PRTHomo sapiens 156Val Glu Leu Glu Val Pro Gln Leu Cys Ser Phe Ile Leu Lys1 5 1015711PRTHomo sapiens 157Ala Pro Glu Pro Asp Leu Met Ser Pro Thr Arg1 5 1015813PRTHomo sapiens 158Glu Val Glu Val Glu Tyr Ser Thr Val Ala Ser Pro Arg1 5 1015914PRTHomo sapiens 159Ser Tyr Gly Ser Val Leu Leu Ser Ala Glu Glu Phe Gln Lys1 5 1016012PRTHomo sapiens 160Asp Ile Asn Asp His Ser Pro Thr Phe Leu Asp Lys1 5 1016110PRTHomo sapiens 161Leu Asn Lys Pro Ala Asp Phe Ile Gly Lys1 5 1016222PRTHomo sapiens 162Phe Asn Pro Ile Cys Lys Ala Ser Gly Trp Pro Leu Pro Thr Asn Glu1 5 10 15Glu Met Thr Leu Val Lys 2016314PRTHomo sapiens 163Val Leu Thr Pro Tyr Cys Tyr Thr Ile Asp Val Glu Ile Lys1 5 1016418PRTHomo sapiens 164Leu Ile Ala Arg Tyr Ala Ala Arg Leu Ala Ala Glu Ala Gly Asn Val1 5 10 15Thr Arg16519PRTHomo sapiens 165Ser Cys Asp Ala Ser Leu Ala Glu Leu Gly Asp Asp Lys Ser Gly Asp1 5 10 15Ala Ser Arg16610PRTHomo sapiens 166Val Tyr Glu Met Tyr Val Thr Val His Arg1 5 1016715PRTHomo sapiens 167Ile Leu Ser Ile Asp Arg Asn Leu Arg Asn Gln Leu Phe Asn Arg1 5 10 1516810PRTHomo sapiens 168Tyr Val Gly Thr Gly Gly Ile Asp Gln Lys1 5 1016910PRTHomo sapiens 169Asn Met Pro Ser Ser Lys Asp Thr Arg Lys1 5 1017010PRTHomo sapiens 170Arg Ala Ile Ile Glu Asp Leu Glu Pro Arg1 5 1017114PRTHomo sapiens 171Lys Asp Ala Phe Leu Tyr Gln Arg Thr Leu Gln Phe Ile Arg1 5 1017218PRTHomo sapiens 172Glu Phe Tyr Ser Glu Ile Leu Asp Lys Lys Phe Thr Val Thr Val Thr1 5 10 15Met Arg17311PRTHomo sapiens 173Trp Leu Ser Ser Pro Val Phe Ser Leu Arg Arg1 5 1017411PRTHomo sapiens 174Ala Val Ile Phe Ile Thr Lys Arg Gly Leu Lys1 5 1017530PRTHomo sapiens 175Val Pro Thr Pro Gln Ala Ile Arg Ala Ala Gln Gly Leu Leu Ala Cys1 5 10 15Gly Val Ala Gln Gly Ala Leu Arg Ser Asn Tyr Val Leu Lys 20 25 3017612PRTHomo sapiens 176Asn Gln Ala Asn Ile His Ala Val Asp Asn Phe Lys1 5 1017710PRTHomo sapiens 177Val Leu Ser Pro Tyr Asp Leu Thr His Lys1 5 1017812PRTHomo sapiens 178Asn Asn Lys Gly Pro Val Lys Val Val Val Gly Lys1 5 101798PRTHomo sapiens 179Arg Gly Ala Asn Val Asn Leu Arg1 518016PRTHomo sapiens 180Ser Glu Gln Pro Thr Leu Val Thr Ala Ser Lys Asp Gly Tyr Phe Lys1 5 10 1518117PRTHomo sapiens 181Val Phe Lys Met Leu Leu Gly Asp Glu Lys Gln Ser Ile Val Gln Ala1 5 10 15Lys18213PRTHomo sapiens 182Gly Ala Asp Pro Pro Pro Pro Pro Ser Arg Thr Gly Arg1 5 1018317PRTHomo sapiens 183Glu Asn Lys Asn Val Lys Asp Pro Val Glu Ile Gln Thr Leu Val Asn1 5 10 15Lys18413PRTHomo sapiens 184Phe Asn Asp Glu Asp Ile Glu Lys Met Glu Gly Lys Lys1 5 1018520PRTHomo sapiens 185Ala Asp Gln Glu Gly Ala Arg Glu Ile Val Cys Asn Val Thr Leu Gly1 5 10 15Gly Glu Arg Arg 2018616PRTHomo sapiens 186Ala Gly Glu Cys Val Thr Ala Gly Gly Leu Gly Gly Ala Arg Arg Arg1 5 10 1518720PRTHomo sapiens 187Gln Val Ser Gln Pro Ala Leu Val Ile Pro Pro Gln Pro Pro Thr Thr1 5 10 15Gly Pro Pro Arg 2018812PRTHomo sapiens 188His Leu Leu Thr Ile Ala Gly Trp Lys His Glu Lys1 5 1018913PRTHomo sapiens 189Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg1 5 1019013PRTHomo sapiens 190Phe Lys Pro Met Gln Arg His Leu Phe Leu His Glu Lys1 5 1019112PRTHomo sapiens 191Val Arg Ala Ser Gln Glu Leu Glu Met Ser Leu Lys1 5 1019216PRTHomo sapiens 192Glu Lys Leu Ser Leu Glu Pro Val Leu Pro Ala Arg Asn Pro Asn Arg1 5 10 1519317PRTHomo sapiens 193Glu Asp Pro Asp Phe Leu Gly Ala Phe Leu Gly Glu Leu Leu Pro Ser1 5 10 15Arg19413PRTHomo sapiens 194Val Met Ser Asp Gly Pro Val Lys Pro Glu Gly Ala Lys1 5 1019518PRTHomo sapiens 195Asp Ser Gly Gly Gln Thr Ser Ala Gly Cys Pro Ser Gly Trp Leu Gly1 5 10 15Thr Arg19611PRTHomo sapiens 196Tyr Leu Ala Ile Val His Pro Leu Lys Pro Arg1 5 1019713PRTHomo sapiens 197Gln Ala Ala Cys Leu Glu Gln Ile Arg Glu Met Glu Lys1 5 1019824PRTHomo sapiens 198Leu Ser Ala Leu Pro Pro Glu Gly Gly Lys Pro Tyr Leu Glu Ser Phe1 5 10 15Lys Leu Ser Leu Asp Asn Thr Lys 201998PRTHomo sapiens 199Gln Thr Val Glu Val Leu Asp Lys1 520011PRTHomo sapiens 200Thr Thr Trp Met Lys Ala Asp Glu Val Glu Lys1 5 1020111PRTHomo sapiens 201Phe Asp Leu Lys Gln Trp Leu Ser Ala Thr Lys1 5 1020217PRTHomo sapiens 202Phe Pro Gly Val Gly Val Leu Pro Gly Val Pro Thr Gly Ala Gly Val1 5 10 15Lys20318PRTHomo sapiens 203Tyr Ala Gln Ile Cys Leu Trp Ile His Gly Asn Asp Ala Asn Cys Ala1 5 10 15Tyr Gly20415PRTHomo sapiens 204Cys Leu Arg Trp Gly Gly Gly Tyr Glu Gly Glu Asn Leu Met Lys1 5 10 1520512PRTHomo sapiens 205Met Ile Met Ile Leu Val Leu Leu Ser Leu Gly Arg1 5 1020613PRTHomo sapiens 206Gly Ser Asp Leu Pro Pro Asp Pro His Asn Cys Gly Lys1 5 102077PRTHomo sapiens 207Ser Leu Leu Glu Glu Glu Arg1 520814PRTHomo sapiens 208Ala Arg Arg Phe Leu Trp Asn Leu Ile Leu Asp Leu Ile Lys1 5 102098PRTHomo sapiens 209Arg Phe Asn Val Ala Ile Thr Arg1 521013PRTHomo sapiens 210Pro Leu Ala Ile Ala Lys Gln Ala Ser Phe Ser Ser Lys1 5 1021115PRTHomo sapiens 211Ser Met Ser Cys Gln Glu Phe Ile Thr Asn Leu Asn Gly Leu Arg1 5 10 1521216PRTHomo sapiens 212Glu Lys Glu Gln Glu Ala Gly Gly Val Ser Tyr Trp Asp Ile Gly Lys1 5 10 1521318PRTHomo sapiens 213Arg Gln Arg Gly Asp Ala Gly Ser Pro Trp Gly Cys Pro Leu Cys Pro1 5 10 15Asp Arg21414PRTHomo sapiens 214Arg Asn Pro Glu Asp Val Gln Glu Ile Ile Val Trp Lys Arg1 5 102158PRTHomo sapiens 215Leu Ala Glu Ser Glu Gly Thr Lys1 521617PRTHomo sapiens 216Phe Leu Asn Leu Gln Asn Glu His Glu Lys Ala Leu Gly Thr Trp Lys1 5 10 15Arg21715PRTHomo sapiens 217Thr Cys Leu Leu Asn Glu Thr Gly Asp Glu Pro Phe Gln Tyr Lys1 5 10 15

Claims

1. A method for detecting breast cancer in a patient, comprising:(i) obtaining a biological sample from said patient; and(ii) evaluating said sample or a fraction of said sample for the presence of at least one biomarker selected from the group of peptides having the amino acid sequences of SEQ ID NOs: 132-217,wherein the presence of said at least one biomarker is indicative of breast cancer.

2. The method according to claim 1, further comprising, prior to the evaluation step, harvesting low molecular weight peptides from said sample to generate at least one fraction comprising said peptides.

3. The method according to claim 1, wherein said biological sample is blood, serum or plasma.

4. The method according to claim 1, wherein the evaluation step comprises an assay selected from the group consisting of mass spectrometry, immunoassay, immuno-mass spectrometry and suspension bead array.

5. The method according to claim 4, wherein said immunoassay is an enzyme linked immunosorbent assay or ELISA.

6. The method according to claim 4, wherein said mass spectrometry comprises multiple reaction monitoring (MRM).

7. The method according to claim 2, further comprising digesting said low molecular weight peptides.

8. The method according to claim 7, wherein said digestion comprises a trypsin digestion.

9. The method according to claim 1, wherein the breast cancer is in an early stage.

10. The method according to claim 1, wherein the breast cancer is in stage T1a.

11. The method according to claim 1, wherein said evaluation step comprises evaluating said sample for the presence of at least biomarkers having the amino acid sequences of SEQ ID NOs: 132, 139, 141 and 148.

12. A method for monitoring the progression of breast cancer in a patient, comprising:(i) obtaining a biological sample from said patient;(ii) evaluating said sample or a fraction of said sample for the presence of at least one biomarker selected from the group of peptides having the amino acid sequences of SEQ ID NOs: 132-217, wherein the presence of said at least one biomarker is indicative of breast cancer; and optionally(iii) repeating steps (i) and (ii).

13. The method according to claim 12 further comprising, prior to the evaluation step, harvesting low molecular weight peptides from said sample to generate at least one fraction comprising said peptides.

14. The method according to claim 12, wherein said evaluation step comprises evaluating said sample for the presence of at least biomarkers having the amino acid sequences of SEQ ID NOs: 132, 139, 141 and 148.

15. An antibody specific for a peptide selected from the group of peptides having the amino acid sequences of SEQ ID NOs: 132-217.

16. The antibody according to claim 15, wherein said antibody is a monoclonal antibody.

17. The antibody according to claim 15, wherein said antibody is a polyclonal antibody.

18. The antibody according to claim 15, wherein said antibody is a chimeric antibody.

19. The antibody according to claim 15, wherein the peptide is selected from the group of peptides having the amino acid sequences of SEQ ID NOs: 132, 139, 141 and 148.

20. A kit for detecting breast cancer in a patient, comprising at least one antibody according to claim 15.

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